One-bead-one-inhibitor-one-substrate screening of neuraminidase activity

Given the eminent threat of a 21st century flu pandemic, the search for novel antiviral compounds is an increasingly important area of research. Recent developments in antiviral research have established the viability of targeting viral neuraminidase (NA), an enzyme that cleaves sialic acid from the cell-surface-mediating passage of the virus in the respiratory tract. N-acetyl neuraminic acid (NeuAc) is the substrate for NA, and analogues of this core structure have been commercialized as antiviral therapeutics. Recent studies have established that this system is well suited for combinatorial approaches to drug discovery. An important step in the process is to develop solid-phase screening technologies. The feasibility of performing competitive solid-phase NA assays is reported herein. Initially, a fluorogenic NeuAc substrate was immobilized on solid support, and the ability of three NAs (Clostridium perfringens, Salmonella typhimurium, and Vibrio cholerae) to cleave the substrate was shown to be analogous to solution-phase assays. The solid support was then bifunctionalized with the fluorogenic NeuAc substrate and one of two known inhibitors (DANA and Zanamivir). The ability of NA to cleave NeuAc from the solid support when simultaneously presented with an inhibitor was shown to be enzyme dependent. As expected, simultaneous presentation of NeuAc and DANA, a nonspecific inhibitor, led to diminished activity for all three enzymes tested. In contrast, dual presentation of NeuAc and the selective inhibitor Zanamivir only showed significant activity against Vibrio cholerae.